Summary of Work: Our research efforts encompassed two general areas: (A) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (B) the instrumental development and applications of vibrational Raman and infrared spectroscopic imaging techniques. (A) Our interest in characterizing the effects of fluctuating lipid microdomains within biomembranes has recently focused on cluster formation within bilayer matrices comprised of lipid mono- or polyunsaturated sn-2 chain and saturated sn-1 chain assemblies. The lateral compressibility properties of these lipid microaggregates are effective in exerting a modulatory influence on induced conformational changes occurring within integral membrane proteins. In studying spectroscopically specific lipid bilayers, appropriate acyl chain deuteration allows the vibrational dynamics of each chain moiety to be monitored separately. Both Raman and infrared spectroscopic techniques were applied toward examining the bilayer series comprised of 1-eicosanoyl(d39)-2-eicosenoyl-sn-glycero-3-phosphocholine [C(20-d39):C(20:1 delta j)PC, with j=5, 8, 11, and 13]. Established order/disorder parameters pertinent to each chain system were assessed as a function of the placement of the unsaturated chain double bond. Both polycrystalline samples and aqueous bilayer dispersions were examined. Various computational techniques provided estimates of chain cluster sizes, which vary between 3-19 acyl chains for this series of phospholipids. For example, the largest sn-1 chain domain, originating specifically from van der Waals interactions between the chains of neighboring molecules, occurs for the C(20-d39):C(20:1 5)PC species. Using these spectroscopic approaches, we have examined in detail the microheterogeneity of a variety of bilayer assemblies. (B)Emphasis has been placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared senstive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. As an example of the utility of the technique in diagnostic pathology, we applied the infrared imaging methodology to a study of cerebellar tissue from mice presenting the morphology and pathology of Niemann-Pick type C disease. The infrared images provided qualitative descriptions of the biochemical differences between unstained tissue from diseased and control animals. The absorbance images, together with their related spectra, allowed the various cellular layers within the tissue to be identified. Statistical analyses of the individual spectra reflecting the various cellular layers provided concise quantitative descriptions of the observed biochemical variations.